The purpose of this proposal is to elucidate the chemical nature, enzymatic properties, metabolic regulation and biological role of a specific gelatinase from human skin. Gelatin degradation is required for further metabolism of the primary products of the collagenases, which catalyze the initial proteolytic cleavage in the collagen molecule. A specific gelatinase has been purified to homogeneity from cultured human skin explants, and isolated in latent form from cultured human skin fibroblasts. This enzyme is a metalloendopeptidase, is heavily blycosylated, and the catalyzes multiple cleavages in whole gelatin chains yielding peptides of greater than 5000 MW. The chemistry of this enzyme will be investigated, particularly with respect to its nature as a glycoprotein. The enzymatic properties of gelatinase - Km, Vmax, cofactor requirements and peptide bond specificity - will also be studied both with whole gelatin and model peptides as substrates. Antibodies to the pure enzyme will be produced, for in vivo localization and quantitation of the enzyme, and for use in a radioimmunoassay for the enzyme. The physiologic regulation of gelatinase will be investigated with particular reference to the questionof whether, in nature, gelatinase is co-regulated with collagenase. Thus, a variety of known effectors of collagenase activity, such a glucocorticoids, cyclic nucleotides, and a number of naturally occurring polypeptide proteinase inhibitors will be examined with respect to their ability to modulate the activity of gelatinase in culture. In conjunction with the foregoing studies, effort will be made to fully purify the gelatinase from cultured human skin fibroblasts. The fibroblast enzyme exists in an inactive form, and, in a partially purified state, very closely resembles the enzyme purified from explant cultures. The availability of the gelatinase in precursor form will greatly facilitate studies in the physiology of gelatinase activation. Lastly, fibroblasts grown from the skin of patients with the inheritied blistering disease dominant epidermolysis bullosa simplex have been found to elaborate markedly reduced amounts of gelatinase activity in culture. The availability of these cells will allow studies on the metabolic consequences of a pathologically diminished ability to degrade gelatin.